1. Introduction
Fibroblast activation protein (FAP, FAP-alpha, seprase, alpha2 antiplasmin converting enzyme) is a Clan SC protease of the prolyl oligopeptidase subfamily S9b, occurring as a cell surface homodimer. FAP has been demonstrated to possess both dipeptidyl peptidase and endopeptidase activity, catalyzed by the same active center. Its expression is associated with activated stromal fibroblasts and pericytes of over 90% of human epithelial tumors examined and with normal or excessive wound healing, e.g. in tissue remodeling sites or during chronic inflammation. The enzyme is generally not expressed in normal adult tissues and in nonmalignant tumors.1 Several studies have tried to map the physiological substrate spectrum of FAP, including very recent reports that identify i.a. alpha2-antiplasmin, type I collagen and gelatin as in vitro substrates of the endopeptidase activity of FAP.2 Analogously, Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY have been identified as in vitro substrates of the dipeptidyl peptidase activity of FAP.3 Nonetheless, the relevance of these findings under in vivo conditions remains debatable and the unambiguous definition of FAP's physiological substrate spectrum remains untouched matter so far.
Through structure-based design studies combined with extensive synthetic and biochemical effort, we were able to establish a Structure-Activity Relationship (SAR) of N-acylated aminoacyl pyrrolidine inhibitors of fibroblast activation protein. This has led to the discovery of a novel scaffold type that has the potential to deliver inhibitors of FAP that combine low nanomolar activity with unprecedented selectivity toward related Clan SC proteases (dipeptidyl peptidases IV, II, 8/9 and the endopeptidase prolyl oligopeptidase (PREP, PO). When compared to most other classes of reported inhibitors of FAP, inhibitors belonging to the scaffold type described here have remarkable stability both in aqueous solution and in human plasma and retain activity and selectivity for FAP within the latter media. For example, WO2007085895, WO2007005991, WO2010083570, WO2006125227 and WO0238590 all disclose FAP inhibitors having a general structure closely relating to the compounds of the present invention. However, none of them actually discloses compounds wherein
as defined in the present invention, is a 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, wherein there are exactly 2 ring atoms between the N atom and X. As further detailed herein below, in particular said feature is relevant for providing the compounds of the present invention with the FAP activity and selectivity as defined herein.
Based on FAP's role in (patho-)fysiology, documented extensively in literature, we reasonably foresee potential applications of our inhibitors in disease domains characterised by: (a) proliferation (including but not limited to cancer) (b) tissue remodelling and/or chronic inflammation (including but not limited to fibrotic disease, wound healing, keloid formation, osteoarthritis, rheumatoid arthritis and related disorders involving cartilage degradation) and (c) endocrinological disorders (including but not limited to disorders of glucose metabolism). The relationship of FAP with said pathological processes is described in more detail hereafter.
(a) FAP and Proliferative Diseases (Including but not Limited to Cancer).
During the last decade, numerous reports have been published that claim an important role for FAP in tumor growth and proliferation. The exact mechanism by which FAP takes part in these processes is unknown, but direct modulation of tumor growth, angiogenesis or disease progression by proteolytic processing of growth factors, cytokines, collagenase activity regulating proteins and even collagen derived proteins, is currently the subject of intensive research.
While awaiting the detailed functional characterization of the enzyme in these processes, several groups currently focus on FAP's status as a potential cancer biomarker which presence or activity in tumors could also be used for site-directed delivery of oncology drugs.4 Equally important, FAP or its activity are being targeted by several groups as a direct way to reduce tumor growth and proliferation by means of immunotherapeutic and small molecule inhibitor approaches.5 For the latter, a number of in vivo proof-of-concept studies are present. These all involve the dipeptide derived boronic acid talabostat (PT-100, Val-boroPro) or close analogues, and report significant activity on tumor stromagenesis and growth.6 In addition, talabostat has been evaluated as a drug in various clinical trials up to phase II, for the treatment of, i.a. metastatic kidney cancer, chronic lymphocytary leukemia, pancreatic adenocarcinoma and non-small cell lung cancer. While talabostat in several of these trials was able to induce clinical response, questions were raised with regards to the safety profile of the compound, potentially related to its well-known lack of selectivity with respect to other Subfamily S9B proteases.7 
(b) FAP and Diseases Involving Tissue Remodeling and/or Chronic Inflammation (Including but not Limited to Fibrotic Disease, Wound Healing, Keloid Formation, Osteoarthritis, Rheumatoid Arthritis and Related Disorders Involving Cartilage Degradation, Atherosclerotic Disease and Chron's Disease)
Multiple reports on occurrence of significantly increased FAP expression and/or activity both in physiological processes and in several clearly distinct disease domains, indicate that the enzyme might play an important role during events characterized by tissue remodeling and/or inflammation. Although the exact mechanism by which FAP is alleged to do so has to date not been clarified, the most straightforward hypothesis involves the enzyme's capability of processing collagenase activity regulating proteins and even collagen derived proteins, thereby altering the composition and structure of the extracellular matrix (ECM) of tissues. This effect could be supplemented by influences on the proteolytic processing of peptide growth factors and cytokines. Similar arguments are summoned to describe the of FAP's role in proliferative disease (vide supra).
Significant FAP expression has been confirmed for reactive fibroblasts in granulation tissue of healing wounds, on stellate cells at the tissue remodeling interface in hepatic cirrhosis, and in lung tissue in idiopathic pulmonary fibrosis.8 For hepatic cirrhosis (the pathological state characterized by fibrosis in which FAP's involvement has been best characterized) elevated expression of FAP was observed regardless of the etiology of the disease (viral hepatitis-induced, alcohol-induced, biliary cirrhosis). This given might suggest broad applicability of FAP-targeted therapy, e.g. using small molecule inhibitors, in disease area's involving fibrotic liver degeneration.9 
FAP expression was found to be significantly increased on keloid fibroblasts compared to normal skin fibroblasts and inhibition of FAP activity with the albeit unselective (with respect to phylogenetically related dipeptidyl peptidases) irreversible inhibitor Gly-Pro(P)(OPh)2 was found to lead to a decrease in invasiveness.10 
FAP expression and activity was also shown to be associated with rheumatoid arthritis and osteoarthritis: FAP-activity on the surface of chondrocytes and elevated expression and activity in cartilage affected by osteoarthritis were demonstrated. FAP was also found to be present in synovial tissue of affected joints, and elevated expression is detected in the murine collagen induced arthritis model. An additional pathway by which FAP could be operating in the pathogenesis and progression of arthritis, has been proposed to imply proteolytic cleavage of alpha2-antiplasmin, ultimately leading to fibrin deposition in the joint. Notably, in a Phase 1 clinical dosing study with a humanized anti-FAP antibody (sibrotuzumab) for advanced and metastatic cancer, the antibody in three patients not only localized to tumors, but also to the knees and shoulders. This observation has been connected to early-stage arthritis, offering initial support for the in vivo validation of FAP as a target for arthritis and related diseases.11 
Recently, significantly increased expression of FAP was reported for human Type IV-Type V aortic atheromata, compared to type III atheromata and healthy aortae. Additionally, thin-cap human coronary atheromata were found to contain more FAP than thick-cap lesions. The enzyme's occurrence was found to be concentrated on smooth muscle and endothelial cells, and it could not be detected on macrophages. Nonetheless, macrophage burden did correlate with total FAP expression in the plaques. Furthermore, in vitro zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed to the enzyme's catalytic domain both in human atherosclerotic smooth muscle cells and in fibrous caps of atherosclerotic plaques.2b 
In a very recent publication, FAP was found to be overexpressed in enteric strictures of patients with Chron's disease (CD) and the protein was observed to be upregulated on strictured CD myofibroblasts by profibrogenic stimuli, leading the authors of this study to propose FAP as a potential target for the treatment of fibrostenosing CD.12 
In general, no in vivo or clinical results (apart from the mentioned) have so far been disclosed dealing with the application of FAP-targeting small molecules or immunotherapeutic strategies in disease domains mentioned under this part. Nonetheless, mounting in vitro evidence from literature can certainly be considered compelling to initiate such investigations.
(c) FAP and Diseases Involving Endocrinological Disorder (Including but not Limited to Disorders of Glucose Metabolism) and Diseases Involving Blood Clotting Disorders.
A recent patent application by Gorrell et al. claims the utility of FAP inhibitors in the prevention and treatment of metabolic abnormalities characterized by abnormal glucose metabolism, including diabetes mellitus and new onset diabetes. This claim is however not otherwise documented in the literature.13 
Finally, blocking the activity of the soluble form of FAP (alpha2-antiplasmin cleaving enzyme, APCE) occurring in plasma, using small molecule inhibitors was found to cause enhanced fibrinolysis and to lead to a decrease of plasminogen activator induced clot lysis time. This observation led the authors to state that APCE-inhibition might constitute a novel approach in thrombolytic therapy without significant risk of bleeding.14 
2. Inhibitor Design
The prime aim underlying our effort to establish detailed SAR data for N-acylated aminoacyl pyrrolidine inhibitors of FAP, was to identify compounds with significantly improved (a) chemical stability and (b) selectivity characteristics when compared to known FAP inhibitors, while retaining high affinity for the target enzyme.
(a) Limited chemical stability due to intramolecular cyclisation is a well known problem of several currently available highly potent dipeptide derived boronic acids (e.g. Val-boroPro). This property, caused by the combined presence of a nucleophilic amino terminus and an electrophilic boronic acid, puts constraints e.g. on the applicability of this compound and its analogues at physiological pH both in vitro and in vivo.15 (b) Selectivity with respect to related S9b proteases (DPP IV, DPP8/9, DPP II, PREP) is a potential point of concern for all FAP inhibitors. Due to the high degree of phylogenetic relationship between the S9b proteases, pharmacophores of their inhibitors generally display substantial overlap. This problem is well documented for a number of described FAP inhibitors, including the well known ValboroPro. Noteworthy however, for most reported FAP inhibitors incomplete and in some cases even no selectivity data have been reported, rendering existing knowledge as a starting point for selective FAP inhibitor discovery inadequate. Nonetheless, taking into account the importance of inhibitor selectivity in the framework of potential compound toxicity and off-target effects, we deemed the preparation of selective compounds an important goal of our endeavours.1 
With the number of reported FAP-inhibitors being small and most of them belonging to the class of boronic acids, we initially decided to focus on compounds that contain a carbonitrile warhead in place of the boronic acid, but conserve an overall dipeptide derived architecture. The latter is a hallmark of most chemotypes of published Subfamily S9B inhibitors. The carbonitrile function itself is also a popular affinity-enhancing moiety in reported series of inhibitors of DPP IV, DPP8, DPP9 and PREP. Compared to other warheads that are used in serine protease inhibitor design (e.g. —B(OH)2, —CHO, chloromethylketones, ketoamides, . . . ) the relatively mildly electrophilic carbonitrile could account for making the inhibitor more selective in vivo, a hypothesis that has been raised in literature earlier.1 In addition, the projected structures' overall architecture does in principle not impose fundamental limitations with respect to in vivo use, as e.g. illustrated by the EMA-approved vildagliptin and the FDA approved saxagliptin, both inhibitors of DPP IV. Three other publications are known to us that also contain carbonitrile-based inhibitors of FAP, all of them including incomplete FAP affinity and selectivity data or, in one case, even no affinity at all.16 
Using the boundary assumptions described above, we decided to start an in depth investigation of the Structure-Activity Relationship (SAR) of N-acylaminoacyl(2-cyanopyrrolidines) as inhibitors of FAP and their selectivity toward dipeptidyl peptidases and PREP. Three main structural fragments within this structure were marked for investigation and elaboration of the SAR:
(a) the P3 Moiety:
By attaching this moiety (via an acyl linkage) to the aminoacyl(2-cyanopyrrolidine) backbone of the inhibitor, we wanted to make the P2 residue non-basic and non-nucleophilic, thus increasing the likeliness of inhibitor selectivity and higher stability with respect to the S9b dipeptidyl peptidases. Some literature evidence existed for peptide derived boronic acid inhibitors that this approach might be viable, although no systematic studies in this direction have been carried out. In addition, a substantial number of these literature FAP inhibitors have been reported with only limited or even without selectivity data for the related dipeptidyl peptidases. Additionally, while one might anticipate affinity toward dipeptidyl peptidases to be smaller, blocking the amino terminus does substantially increase the risk of selectivity problems with respect to the endopeptidase PREP. Again, very limited literature information was present dealing with FAP to PREP selectivity of inhibitors with an acylated P2 amine function.1 
(b) the P2 Moiety:
while several acylated glycyl(2-borono)pyrrolidines have been reported in literature, almost no data exist on the influence of other amino acid residues at the P2 position in acylated compounds. At the outset of our activities, substrate kinetics studies nonetheless indicated a rather strict preference of FAP for a P2-glycine residue in substrates containing an acylated P2 amino function. This given is in sharp contrast with a series of dipeptide-derived substrates and/or inhibitors (e.g. ValboroPro) with a free amino terminus, where the number of tolerated P2 residues is known to be much larger.
(c) The P1 Moiety:
We decided to investigate the influence on activity and selectivity of substituting the pyrrolidine ring in compounds with structure 1. To this end, we selected a number of different functional groups with different bulk size and electronic effects.
In addition, we expected the obtained SAR-information poised to be applicable to analogous inhibitor types containing specific other warhead types or even no warhead, a hypothesis that we later on showed to be correct.
We have now surprisingly found that FAP-inhibitors of formula I exhibit good chemical stability and high selectivity for FAP, rendering them very suitable for the preparation of a medicine for the treatment of various FAP-related disorders. In addition, our invention has the potential to deliver compounds with high solubility and low Log D-values, a feature that is far from evident for dipeptide-derived compounds lacking a basic amino terminus and that is accounted for by the presence of heteroatoms introduced at specific positions of the P3 substituent.